Abnormal activity of dopamine (DA) neurons in the ventral tegmental area (VTA) has been suggested to play a key role in disorders including schizophrenia, drug addiction, and attention deficit hyperactivity disorder (ADHD). Evidence further suggests that the activity of VTA DA cells is under the influence of the prefrontal cortex (PFC), an area critically involved in the brain's executive functions including decision making and impulse control. However, due to the presence of multiple pathways between the PFC and VTA and due to the lack of selectivity of previous techniques, our understanding of how the PFC regulates DA cells remains limited. The goal of this project is to use recently developed optogenetic techniques to selectively activate or inhibit individual PFC-VTA pathways and then to study their effects on DA cells. The key hypothesis to be tested is that the PFC regulates DA neurons through both its direct and indirect projections to the VTA. However, due to the difference in synapses involved, different PFC-VTA pathways may conduct signals in different ways. To test these possibilities, two series of studies will be conducted. The aim of the first series is to express the excitatory channelrodopsin-2 (ChR2) in PFC neurons based on their projection site and then to test whether optical activation of different PFC-VTA pathways produces different effects on DA cells. To express ChR2 in PFC neurons projecting to the VTA, two adeno-associated viral (AAV) vectors will be used: a cre-dependent AAV carrying the ChR2 gene and an AAV encoding WGA-Cre fusion protein. Viruses containing the former will be injected into the PFC and those containing the latter into the VTA. Both in vivo and in vitro recordings will then be used to determine whether optical stimulation of the pathway alters the activity of DA cells. It is predicted that low-frequency stimulation excites DA cells projecting back to the PFC and produces no effect or an inhibition of DA cells projecting the nucleus accumbens (NAc). When stimulated at high frequencies, however, PFC terminals may excite NAc-projecting DA cells via volume transmission. To determine if the PFC also regulates DA cells indirectly, similar methods will be used to express ChR2 and to activate PFC neurons projecting to areas such as the NAc, pedunculopontine tegmental nucleus (PPT), and lateral hypothalamus (LH). The aim of the second series of studies is to understand the role of PFC-VTA pathways in the generation of firing patterns of DA neurons. Previous studies have shown that most spontaneously active VTA DA neurons display a slow oscillation (SO) in firing rate or rhythmic bursting. The activity is at least partially derived from the PFC since it is highly correlated with PFC local field potentials and is inhibited when PFC inputs are blocked. Further analysis suggests, however, that the SO is transferred to DA cells indirectly via inhibitory neurons. To identify the inhibitory pathway, the inhibitory halorhodopsin eNpHR will be expressed in PFC cells projecting to the VTA or NAc. If GABA neurons in the two areas relay the SO to DA cells, inactivation of the two pathways would reduce or eliminate the SO. PFC projections to the PPT and LH will be similarly studied to determine whether they influence only the baseline firing rate and are not required for the SO in most DA cells. The results of the above studies will provide a basis for further investigation of PFC control of DA neurons in schizophrenia, drug addiction, and ADHD.